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How to make a midbrain dopaminergic neuronErnest Arenas1,*, Mark Denham1,2 and J. Carlos Villaescusa1,3

ABSTRACTMidbrain dopaminergic (mDA) neuron development has been anintense area of research during recent years. This is due in part to agrowing interest in regenerative medicine and the hope that treatmentfor diseases affecting mDA neurons, such as Parkinson’s disease(PD), might be facilitated by a better understanding of how theseneurons are specified, differentiated and maintained in vivo. Thisknowledge might help to instruct efforts to generate mDA neuronsin vitro, which holds promise not only for cell replacement therapy, butalso for disease modeling and drug discovery. In this Primer, we willfocus on recent developments in understanding the molecularmechanisms that regulate the development of mDA neurons in vivo,and how they have been used to generate human mDA neuronsin vitro from pluripotent stem cells or from somatic cells via directreprogramming. Current challenges and future avenues in thedevelopment of a regenerative medicine for PD will be identifiedand discussed.

KEY WORDS: Dopamine neurons, Midbrain, Parkinson’s disease,Regeneration, Reprogramming, Stem cells

IntroductionDopaminergic (DA) neurons are capable of releasing dopamine, acatecholaminergic neurotransmitter. They are characterized by thepresence of tyrosine hydroxylase (TH), the rate-limiting enzyme inthe synthesis of catecholamines, and are found throughout themammalian central nervous system, including the ventral midbrain(VM) (Björklund and Hökfelt, 1983). Midbrain DA (mDA)neurons are arranged in three distinct nuclei: the substantia nigrapars compacta (SNc, also known as the A9 group), the ventraltegmental area (VTA, or A10 group) and the retrorubral field (RrF,or A8 group) (Björklund and Hökfelt, 1983; Dahlstroem and Fuxe,1964) (Fig. 1A). Different populations of mDA neurons projectto distinct areas and control or modulate specific functions,according to their targets [reviewed by Roeper (2013)]. VTA andRrF DA neurons project to the ventromedial striatum (nucleusaccumbens), parts of the limbic system and prefrontal cortex,forming the mesolimbic and mesocortical systems (Fig. 1B).These neurons regulate emotional behavior, natural motivation,reward and cognitive function, and are primarily implicated in arange of psychiatric disorders (Carlsson, 2001; Chao and Nestler,2004; Hornykiewicz, 1978). By contrast, DA neurons located inthe SNc primarily project to the caudate-putamen, the dorsolateral

striatum, forming the so-called nigrostriatal pathway, whichpredominantly regulates motor function and degenerates inParkinson’s disease (PD) (Box 1; Fig. 1A and B, in pink). Thisdisorder was first described clinically by the British physicianJames Parkinson in ‘An Essay of the Shaking Palsy’ published in1817 (Horowski et al., 1995). However, it was only many yearslater that the pathological basis for PD was found to relate to thedegeneration of SNc neurons (Foix and Nicolesco, 1925; Graham,1979; Hassler, 1938) and the loss of dopaminergic innervation ofthe striatum (Lloyd and Hornykiewicz, 1970). We now know thatPD involves the degeneration of multiple neuronal subtypesbesides SNc neurons (Jellinger, 1991). However, the cells that aremost affected and responsible for many of the motor features inPD are mDA neurons of the SNc, a cell type that has become aprimary target for cell replacement therapy (Box 1). Our ability togenerate subtype-specific human mDA neurons in vitro mighttherefore hold the key for the development of future regenerativemedicine for PD.

Transplantation of human fetal midbrain tissue in open-labelclinical trials, in which both the researcher and the test subject knowwhat treatment is administered, has provided proof of concept forcell replacement therapy in PD (Arenas, 2010; Evans et al., 2012;Hallett et al., 2014; Lindvall and Björklund, 2004, 2011). However,logistical and ethical difficulties in performing such studies usinghuman fetal tissue have led to the search for more amenable cellpreparations that could be standardized for quality, safety andfunctionality, prior to clinical use in PD. Human mDA neurons havebeen generated from multiple cell types in vitro, includingneural stem/progenitor cells (NS/PCs) (Maciaczyk et al., 2008;Riaz et al., 2002; Ribeiro et al., 2012; Sanchez-Pernaute et al.,2001), pluripotent stem cells (PSCs) (Denham et al., 2012; Grealishet al., 2014; Kirkeby et al., 2012; Kriks et al., 2011; Xi et al., 2012)and by lineage reprogramming of somatic cells such as fibroblasts(Caiazzo et al., 2011; Kim et al., 2011; Pfisterer et al., 2011). Thein vivo functional capacity of PSC-derived mDA cells has recentlybeen found to match that of fetal tissue (Grealish et al., 2014).Achieving cell preparations enriched for fully functional humanSNc DA neurons in vitro and capable of selectively re-innervatingthe dorsal striatum or reconstructing the nigrostriatal pathway is thusthe next challenge. In order to take advantage of the therapeuticpotential that stem cells and reprogramming technologies currentlyoffer, we must improve our knowledge of the molecularmechanisms that control mDA neuron development and use thisto guide strategies to generate SNc DA neurons and improvefunctionality in vivo.

In this Primer article, we discuss recent advances inunderstanding mDA neuron development in vivo, and howthese developmental principles have and continue to influence thedevelopment of stem cell therapies. We also outline the challengesand opportunities that lie ahead in order to efficiently generate safeand functional human mDA neuron preparations for cellreplacement therapy, as well as for disease modeling and drugdiscovery.

1Laboratory of Molecular Neurobiology, Dept. Medical Biochemistry andBiophysics, Center of Developmental Biology for Regenerative Medicine,Karolinska Institutet, Stockholm 171 77, Sweden. 2Danish Research Institute ofTranslational Neuroscience, Nordic EMBL Partnership for Molecular Medicine,Aarhus University, Aarhus 8000, Denmark. 3Institute of Experimental Biology,Faculty of Science, Masaryk University, Brno 61137, Czech Republic.

*Author for correspondence (ernest.arenas@ki.se)

This is an Open Access article distributed under the terms of the Creative Commons AttributionLicense (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,distribution and reproduction in any medium provided that the original work is properly attributed.

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mDA neuron developmentDuring gastrulation, a posterior-to-anterior migration of cellstakes place at the same time as the three germ layers (mesoderm,endoderm and ectoderm) are formed. At the rostral end of the embryo,the inhibitors DKK1 (dickkopf 1, a WNT inhibitor), NOG (noggin, aBMP inhibitor), LEFTY1 (left-right determination factor 1, aNODAL inhibitor) and CERBERUS (a multifunctional inhibitor ofWnt, BMP and Nodal), which suppress posterior signals and patternthe neural ectoderm, leading to the formation of the anterior neuraltube (reviewed by Takaoka et al., 2007). As the development of theneural tube proceeds, two signaling centers are formed: the isthmicorganizer (IsO), which defines the midbrain-hindbrain boundary(MHB) (Joyner et al., 2000; Rhinn et al., 1998; Wassarman et al.,1997), and the floor plate (FP), which controls ventral identities(Placzek and Briscoe, 2005) (Fig. 1C). During these and subsequentdevelopmental stages, the combined action of transcription factors andmorphogens from the IsO and the FP orchestrate multiple functions,including the regional identity of the VM, the specification andproliferation of mDA progenitors, mDA neurogenesis, as well as thedifferentiation and survival of mDA neurons.

Transcriptional regulation of VM patterningOne of the earliest and most crucial patterning events in the neuraltube is the formation of the IsO at the MHB, which starts at E7.5

through the coordinated expression and mutual repression of thetranscription factors,Otx2 (orthodenticle homolog 2) in the midbrainand Gbx2 (gastrulation brain homeobox 2) in the hindbrain,(Fig. 1C) (Broccoli et al., 1999; Millet et al., 1999; Wassarmanet al., 1997). Otx2 and Gbx2 control the patterning of the MHB byregulating the expression of two morphogens,Wnt1 (wingless-int1;wingless-type MMTV integration site family, member 1 – MouseGenome Database) in the midbrain and Fgf8 (fibroblast growthfactor 8) in the hindbrain (Joyner et al., 2000; Rhinn et al., 1998)(Fig. 1D and Fig. 2, yellow area). Whereas Otx2 is required forthe expression of Wnt1 (Puelles et al., 2004; Rhinn et al., 1999),the induction of Fgf8 requires Pax2 (paired homeobox 2) and ismaintained by Gbx2 (Ye et al., 1998). Importantly, Fgf8, but notWnt1, is required (Chi et al., 2003) and sufficient (Martinez et al.,1999) for the induction of the IsO. Accordingly, cells atthe MHB acquire anteroposterior information by interpreting theconcentration gradient generated by the secretion of FGF8 inthe IsO. Whereas high concentrations of FGF8 in the hindbraindrive a hindbrain cell fate, lower concentrations in the neighboringanterior tissue ensure that the cells adopt a midbrain identity(Basson et al., 2008; Sato and Nakamura, 2004; Suzuki-Hiranoet al., 2005). In agreement with this, FGF8 can induce mDA neuronsin brain explants, at a certain distance from the FGF8 source (Yeet al., 1998), by a process that requires Wnt1 (Prakash et al., 2006)

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Fig. 1. Distribution of mDA neurons, their projections in the adult mouse brain and the expression of genes important for their development. (A) Coronalsection of the adult brain at the midbrain level, showing the position of the three mDA nuclei: RrF/A8, SNc/A9 and VTA/A10. (B) Sagittal view of the adult brain witha schematic representation of mDA neurons and their projection areas. A8, retrorubral; A9, substantia nigra; A10, ventral tegmental area; A8+A10, structuresinnervated by A8 and A10; A8-A10, structures innervated by A8, A9 and A10; ACC, anterior cingulate cortex; CNA, central nucleus of the amygdala; DS, dorsalstriatum; ERC, entorhinal cortex; H, habenula; HC, hippocampus; LC, locus coeruleus; LS, lateral septum; NA, nucleus accumbens; OT, olfactory tubercle;PFC, prefrontal cortex; PRC, perirhinal cortex; PC, pyriform cortex. (C,D) Sagittal view of an E11.5 mouse embryo, showing the expression of transcription factorsand morphogens important for VM patterning in relation to Th expression. D, diencephalon; H, hindbrain; IsO, isthmic organizer; M, midbrain; T, telencephalon.(E) Coronal section through the VM at the level shown by arrows in D. BP, basal plate; mFP, midbrain floor plate.

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(Fig. 2, yellow and blue). Notably, Wnt1 expression at the MHB isrequired for the development of both posterior midbrain and anteriorhindbrain (McMahon and Bradley, 1990; McMahon et al., 1992;Thomas and Capecchi, 1990), but its anterior expression in twoparamedial bands within the midbrain FP (Fig. 1E) serves anadditional, crucial role in the specification and differentiation ofmDA progenitors [reviewed by Arenas (2014)].A second major event is the patterning of the neural tube by the

morphogen SHH (sonic hedgehog) (Roelink et al., 1995). SHH,initially secreted by the notochord specifies the most ventral regionof the neural plate, the FP, by inducing the expression of Foxa2 atE8 in mice (Ang et al., 1993; Sasaki et al., 1997). Foxa2 plays acentral role in the SHH network and is required for notochord andFP development as well as for ventral patterning (Ang and Rossant,1994;Weinstein et al., 1994). By E8.5, the FP itself starts expressingShh and becomes a secondary ventral organizer of the neuraltube (Fig. 1C-E). A gradient of SHH from the FP regulatespatterning in the ventro-dorsal axis: ventral progenitors exposed tohigher concentrations of SHH respond by expressing differenttranscription factors compared with dorsal cells, exposed to lowerconcentrations, resulting in different ventro-dorsal identities(Briscoe and Ericson, 1999; Chiang et al., 1996; Ericson et al.,1996; Marti et al., 1995; Roelink et al., 1995). SHH regulatestranscription by binding to its receptor PTCH1 (patched 1), whichthen releases its inhibition on a co-receptor, SMO (smoothened),that signals to prevent the processing of the GLI (glioma-associatedoncogene) transcription factors GLI1 and GLI2 into repressors.

Thus, Shh biases the balance of GLI processing towards anactivated state, leading to the upregulation of ventrally expressedhomeodomain transcription factors. In the midbrain FP, high SHHsignaling upregulates Foxa2 (Sasaki et al., 1997) (Fig. 2, pink area),whereas, in the basal plate (BP), a more lateral and dorsal positionflanking the FP, lower levels of SHH upregulate Nkx6-1 and Otx2(Nkx for NK homeobox protein) (Fig. 2, green area). FOXA2 alsoinduces Nkx6-1 in the BP (Nakatani et al., 2010) and suppressesNkx2-2 in the midbrain FP (mFP) (Ferri et al., 2007). Moreover,FOXA2 directly represses Gli1-3 and upregulates Shh expression(Metzakopian et al., 2012). Overall, these studies indicate thatventral patterning involves both a temporal and spatial responses toSHH signaling.

Specification of mDA progenitors and suppression of lateral fatesFate mapping experiments have indicated that mDA neuronsoriginate from progenitors sorted for the FP marker CORIN (Onoet al., 2007) or expressing the radial glia marker Glast/Slc1a (glialhigh affinity glutamate transporter) (Bonilla et al., 2008). Thisshows that mFP radial glial cells, unlike FP radial glia in other brainregions, can undergo neurogenesis and are mDA progenitors.Additionally, mDA neurons have been fate-mapped fromprogenitors expressing Shh (Blaess et al., 2011; Hayes et al.,2011; Joksimovic et al., 2009a) or Wnt1 (Brown et al., 2011; Yanget al., 2013a; Zervas et al., 2004), indicating that mFP radial glia arealso constituents of the two main signaling centers, the mFP and theIsO. Moreover, information from both signaling centers isintegrated in mDA progenitors, in which FOXA1/2 (Lin et al.,2009) and OTX2 (Ono et al., 2007) are present (Fig. 3) and regulatethe expression of two LIM homeobox transcription factors, Lmx1band Lmx1a (Fig. 2, blue area). Whereas Lmx1b is necessary for thedifferentiation of mDA progenitors (Smidt et al., 2000), Lmx1a isrequired for the specification of mDA neurons in the mFP(Andersson et al., 2006b; Deng et al., 2011) and, via Msx1(muscle segment homeobox homolog 1), to suppress the emergenceof BP fates (Andersson et al., 2006a). Additionally, Otx2 sustainsthe expression Nkx6-1 in the BP and suppresses Nkx2-2 in the mFP(Puelles et al., 2004). Thus, the concerted action of the Shh-Foxa2and the Otx2-Wnt1-Lmx1a/Msx1 networks is essential not only forthe specification of the mFP but also for the suppression ofalternative neural fates (Fig. 2, green area).

In other parts of the neural tube,Wnt1/β-catenin (Ctnnb),Lmx1a andLmx1b are typical dorsal roof plate genes (Millonig et al., 2000;Shimamura et al., 1994). However, in the mFP, they form a positiveautoregulatory loop (Chung et al., 2009) (Fig. 2, red arrows) requiredfor mDA specification: on one hand β-catenin directly upregulatesLmx1a and Otx2, and, on the other hand, Lmx1a/b directly upregulateeach other as well as Wnt1, Msx1 and two key genes involvedin mDA neuronal differentiation and survival, Nurr1 (Nr4a2,nuclear receptor 4a2) and Pitx3 (pituitary homeobox 3, or paired-like homeodomain transcription factor 3). Accordingly, deletion ofboth Lmx1a and Lmx1b results in a near-complete loss of mDAneurons (Yan et al., 2011). Similarly, deletion of Wnt1 results in theloss of Lmx1a, Nurr1 and Pitx3 in the mFP and a complete loss ofmDA neurons (Andersson et al., 2013; Prakash et al., 2006).Combined, these results indicate that the specification of mDAneurons is controlled by the Lmx1a/b-Wnt1/Ctnnb autoregulatory looptogether with Otx1/2 and Foxa1/2.

mDA neurogenesismDA neurogenesis takes place in the ventricular zone (VZ) of themFP, when mDA progenitors divide to generate postmitotic cells

Box 1. Parkinson’s disease and its treatmentParkinson’s disease (PD) is a progressive neurodegenerative disorder,characterized by the loss of mDA neurons of the SNc that project to thestriatum (Fig. 1A,B). These neurons control motor behavior, and, as theydegenerate, they result in several motor features of the disease, such asbradykinesia, rigidity, resting tremor, gait disturbances and posturalinstability (Lees et al., 2009). However, other cell types are alsoprogressively affected in PD, such as locus coeruleus noradrenergicneurons (Hassler, 1938), basal forebrain cholinergic neurons (Tagliaviniet al., 1984), peptidergic neurons (Agid et al., 1986) and serotoninneurons (Calabresi et al., 2013; Chaudhuri et al., 2006; Chaudhuri andSchapira, 2009; Fox et al., 2008; Halliday et al., 2011). The loss of theseneurons results in non-motor features, including cognitive, affective,sleep, olfactory and intestinal alterations (Chaudhuri et al., 2006;Chaudhuri and Schapira, 2009).

The main treatment for PD is pharmacological. The most efficient drugis the dopamine precursor levodopa (Brichta et al., 2013), but otheragents, including dopamine agonists, catechol-o-methyl-transferase(COMT) and monoamine oxidase type B (MAOB) inhibitors, as well asnon-dopaminergic agents, such as antidepressants or cholinesteraseinhibitors for dementia, are also prescribed (Connolly and Lang, 2014).The chronic use of levodopa is associated with motor complications,including fluctuations and dyskinesias (Stern et al., 2004; Weiner, 2004),whereas dopamine agonists can cause behavioral alterations (Jankovicand Aguilar, 2008). Deep brain stimulation (DBS), targeting the thalamus,subthalamic nucleus and globus pallidus, is currently used in PD patientswhose motor symptoms cannot be adequately controlled by medication.The mechanism of action is not known, but DBS is thought to blockdepolarization, activate inhibitory neurons, desynchronize tremorogenicpacemakers and functionally disrupt neuronal networks (Jankovic andPoewe, 2012).

Although all these treatments relieve some symptoms of PD, they donot slow down disease progression or reverse the damage to mDAneurons, and the treatment loses efficacy. Cell replacement therapy hasthus gained interest as a therapeutic option, as it has the potential tochange the course of disease.

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which express the nuclear receptor Nurr1. These cells migratethrough the intermediate zone (IZ) while they differentiate andbecome TH+ mDA neurons on reaching the mantle zone (MZ)(Fig. 3A). In rodents, mDA cells appear at E10.5, whereas, inhumans, mDA neurogenesis begins between 5.0 and 6.0 weeks postconception (PC), peaks at weeks 6-8 (Almqvist et al., 1996;

Nelander et al., 2009) and ceases at weeks 10-11 (Freeman et al.,1991). mDA neurogenesis is controlled by two proneural genes ofthe basic-helix-loop-helix family, Mash1 (mouse achaete-schutehomolog 1) andNgn2 (Neurog2; neurogenin 2) (Fig. 2, purple area),expressed in the VZ of the mFP. Ngn2 is required for mDAneurogenesis (Andersson et al., 2006a; Kele et al., 2006), whereas

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Fig. 2. Genetic networks controlling the development of the midbrain-hindbrain and mDA neurons in the mouse brain. Antero-posterior patterning(yellow area): The mutually repressive activities of Otx2 in the midbrain (MB) and Gbx2 in the hindbrain (HB) establish the midbrain-hindbrain boundary (MHB),where Otx2 is inhibited in the hindbrain (HBi) and Gbx2 in the midbrain (MBi). Midbrain floor plate (mFP) specification (blue area): LMX1B expression in themidbrain mFP directly regulates the expression of Wnt1 and Lmx1a, which also regulate each other via CTNNB (bCat) or directly (LMX1A), forming anauto-regulatory loop (shown by red arrows).Wnt1 regulates Otx2 and Lmx1a via β-catenin, and Lmx1a/b regulateMsx1 (Wnt1-Lmx1a/b-Msx1 network). Ventralpatterning (pink area): FOXA2 regulates Shh, which feeds back onto Foxa2 via GLI (Shh-Foxa2 network). FOXA2 also directly regulates Lmx1a/b, to coordinatethe specification of mDA neurons. Lateral phenotypes (green area): Midbrain basal plate (mBP) markers (Nkx2-2 and Nkx6-1) are inhibited in the mFP (FPi)by Foxa2 andOtx2 (Nkx2-2), and byMsx1 (Nkx6-1).Neurogenesis (purple area): The expression ofNgn2 is indirectly regulated byWnt5a, Foxa2 (via Ferd3l andHes1), Lmx1a/b (viaMsx1) and Lxr alpha/beta (Nr1h3/Nr1h2).Wnt1/bCat andOtx2 regulate both Ngn2 andMash1. mDA neuroblasts and neurons (beige area):LMX1A directly regulates the expression of mDA postmitotic genes, such asNurr1 andPitx3, which in turn regulate Th. These postmitotic genes are also regulatedby Foxa2, Ngn2, Wnt5a, Lxr alpha/beta, Wnt1/β-catenin and Otx2. Solid lines indicate direct interactions as demonstrated by chromatin immunoprecipitation.All other interactions, whether direct or indirect, are shown by dashed lines. Arrowheads indicate activation and perpendicular lines denote inhibition.

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Fig. 3. Gene expression in themDA lineage. (A) Schematic representation of a section through the mFP at E11.5. The ventricular zone (VZ) contains radial gliacells (RG, blue) that undergo neurogenesis to generate postmitotic neuroblasts (yellow) that migrate radially through the intermediate zone (IZ), over theprocesses of the RG and differentiate into mDA neurons (red) in the marginal zone (MZ). As cells become mDA neurons, they migrate tangentially towards thesubstantia nigra pars compacta (SNc). Arrows indicate radial migration (RM) of neuroblasts and tangential migration (TM) of mDA neurons. (B) RG (blue) cellsexpress morphogens, proneural and early transcription factors, some of which are also expressed in neuroblasts (Nb, yellow) and mDA neurons, defining theentire mDA lineage. ±, low levels of expression; +, expressed; A, mainly anterior midbrain; P, mainly posterior midbrain.

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Mash1 is capable of partially compensating for the loss of Ngn2(Kele et al., 2006). These two proneural genes are directly orindirectly regulated by, and integrate information from, the Shh-Foxa2 and Lmx1a/b-Wnt1-Otx2 networks (Fig. 2, pink, blue andyellow areas), as well as nuclear receptors of the Liver X receptorfamily (Lxrα/Nr1h3 and Lxrβ/Nr1h2) (Sacchetti et al., 2009) andthe morphogen Wnt5a (Andersson et al., 2008) in order to controlmDA neurogenesis (Fig. 2, purple area).Repression of Shh, but not Foxa2, by Wnt1/Ctnnb in the mFP is

required for mDA neurogenesis (Andersson et al., 2013; Joksimovicet al., 2009b; Tang et al., 2009). Foxa2 then, together with Foxa1,dose-dependently controls mDA neurogenesis (Ferri et al., 2007;Stott et al., 2013) in twoways, by directly regulating the basic helix-loop-helix gene Ferd3l (Fer3-like, also known asNato3) and Lmx1a(via Msx1) (Metzakopian et al., 2012). Whereas Ferd3l regulatesmDA neurogenesis by repressing Hes1 (hairy and enhancer ofSplit 1) (Ono et al., 2010), a suppressor of pro-neural genes, Lmx1aincreases the expression ofNgn2 viaMsx1 (Andersson et al., 2006b)(Fig. 2, purple area).However, CTNNB also regulates mDA neurogenesis by directly

regulating the expression of Lmx1a and Otx2 (Chung et al., 2009),and Otx2 is required for mDA neurogenesis by controlling theexpression of Ngn2 andMash1 (Omodei et al., 2008; Prakash et al.,2006). Similarly, Wnt1 is required for the expression of Ngn2 andMash1 in the mFP and for mDA neurogenesis (Andersson et al.,2008). Another important factor for mDA neurogenesis is themorphogen Wnt5a, which is expressed in the VM at E9.5 andbecomes restricted to the BPand themFP byE11.5-13.5 (Fig. 1D,E).Wnt5a is expressed in mFP radial glia, neuroblasts and partially inposterior mDA neurons (Fig. 3B), where it inhibitsFoxa2,Ngn2 andNurr1 expression (Andersson et al., 2008) (Fig. 2, purple area).Accordingly, deletion ofWnt5a does not impair mDA neurogenesis,but rather increases the number of postmitotic NURR1+/TH− cells(Andersson et al., 2008). Surprisingly, however, its deletionpotentiates the neurogenesis defect in Wnt1−/− mice, indicatingthat there is cooperation between both Wnts in mDA neurogenesis(Andersson et al., 2013).LXRα and LXRβ are two additional regulators of mDA

neurogenesis. These nuclear receptors are ligand-dependenttranscription factors that form obligate heterodimers with RXR(retinoid X receptors). LXRs are required to maintain the expressionlevels of Lmx1b, Wnt1 and Ngn2 in the developing midbrain, anddeletion of both Lxr receptors decreased mDA neurogenesis,whereas their overexpression increased it (Sacchetti et al., 2009)(Fig. 2, purple area). Moreover, activation of LXRs by theendogenous midbrain LXR ligand 24,25-epoxycholesterol hasbeen shown to selectively and specifically promote mDAneurogenesis, in an LXR-dependent manner (Theofilopouloset al., 2013). These results indicate that LXRs are both requiredand sufficient for mDA neurogenesis during development and thatLXR ligands constitute a new class of selective and cell type-specific regulators of neurogenesis (Theofilopoulos et al., 2013).

Migration of postmitotic mDA neuroblasts and neuronsAfter neurogenesis, postmitotic cells migrate through the IZ towardstheir final destination in the MZ (Hanaway et al., 1971; Kawanoet al., 1995). mDA cells first migrate radially along the vimentin+

radial glial processes (Shults et al., 1990) (Fig. 3A) and thentangentially, to reach their final position in the SN, VTA and RrF(Hanaway et al., 1971; Marchand and Poirier, 1983) (Fig. 1A).Cxcr4 (C-X-C motif chemokine receptor type 4) can be detected inthe IZ and MZ (Fig. 3B) and is required for radial migration andfiber outgrowth of mDA neurons between E11.5 and E14.5 (Bodeaet al., 2014; Yang et al., 2013b). Its ligand, Cxcl12 (C-X-C motifchemokine 12) is expressed in the meninges and is sufficient topromote the migration of mDA neurons (Yang et al., 2013b).Whereas pharmacological inhibition or deletion of Cxcr4 does notaffect the number of mDA neurons, it affects their migration, assome cells remain in the IZ and do not reach the MZ (Yang et al.,2013b). Tangential migration of mDA neurons is regulated by theneural L1 cell adhesion molecule (L1CAM) (Demyanenko et al.,2001; Ohyama et al., 1998) and RELN (reelin) (Kang et al., 2010;Nishikawa et al., 2003). Mice lacking Reln or the cytoplasmicadaptor protein Dab1 (disabled 1) have fewer PSA-NCAM+

tangential fibers and fewer mDA neurons reaching the SNc,despite normal numbers of mDA neurons being generated (Kanget al., 2010). Moreover, the RELN receptors LRP8 (low densitylipoprotein receptor-related protein 8, also known as ApoER2) andVLDLR (very low density lipoprotein receptor) are also required forthe migration and final positioning of mDA neurons (Sharaf et al.,2013). Finally, Ntn1 (netrin 1) regulates mDA neuron migration inthe mFP (Livesey and Hunt, 1997), and deletion of the NTN1receptor Dcc (deleted in colorectal cancer), results in not onlydorsally displaced mDA neurons, but also in a loss of mDA neuronsand aberrant innervation of nigrostriatal and mesocortical targets(Flores et al., 2005; Riedl and Salvesen, 2007; Xu et al., 2010).Thus, multiple pathways regulate mDA neuron migration – mostnotably CXCL12/CXCR4 to control radial migration, and RELNsignaling to control tangential migration.

Differentiation and survival of mDA neuronsAfter neurogenesis, migratory postmitotic neuroblasts in the IZdifferentiate into mDA neurons in the MZ. This process is regulatedby some of the early factors described above, such asOtx2,Lmx1a/b,Foxa1/2 and the homeobox genes En1/2 (engrailed 1/2), whichremain expressed in postmitotic mDA cells (Fig. 3B). Early factorsin turn regulate the activity of late transcription factors, such asNurr1and Pitx3 (Fig. 2, beige, Fig. 3B), which control the progressiveacquisition of appropriate neurotrophic factor and DAneurotransmitter phenotype (Table 1).

The morphogen WNT5A also promotes the differentiation ofrodent and humanmidbrain progenitors into functionalmDAneurons(Andersson et al., 2008; Parish et al., 2008; Ribeiro et al., 2012).Wnt5a−/− mice show an excess of NURR1+;TH− postmitotic DAneuroblasts but not mDA neurons, indicating a differentiation defect.

Table 1. Late transcription factor genes, such as Nurr1, En1 and Pitx3, regulate the expression of multiple genes in mDA neurons

TFs mDA NFs Post

Nurr1 En1 Pitx3 Aadc/Ddc Ahd2 TH Vmat Dat Drd2 Bdnf C-ret Cck Nts Dlk1

Nurr1 + + + + + + + + + + +En1 + + + + + + +Pitx3 + − + + + + + + − − −

Note that Pitx3 is highly expressed in the anterolateral mDA neurons, where it inhibits the expression of en1 and posterior markers such as cck. +, positiveregulation; −, negative regulation. TFs, transcription factors; NFs, neurotrophic factors; Post, posterior markers.

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Wnt5a−/− mice display typical convergent-extension morphogenicdefects, resulting in a rostro-caudal shortening and medial-lateralwidening of the mDA domain (Andersson et al., 2008). Deletion ofWnt1 does not affect conversion-extension and, unlike Wnt5a,decreases the number of NURR1+ neuroblasts and TH+ mDAneurons (Andersson et al., 2013). However, simultaneous deletion ofWnt5a andWnt1 further enhances theWnt1−/−phenotype and reducesthe number of NURR1+ and TH+ mDA neurons to a greater extent,indicating a cooperation between these two Wnts in mDAdifferentiation (Andersson et al., 2013) (Fig. 1D,E). Wnt1 is alsorequired for the survival of mDA neurons and the expression of Pitx3(Prakash et al., 2006) aswell as LMX1A in themFP (Andersson et al.,2013), where LMX1A/B directly regulates Nurr1 and Pitx3 (Chunget al., 2009). However, FOXA1/2 are required for the expression ofNurr1, En1 and Ddc (dopa decarboxylase, also known as aromaticL-amino acid decarboxylase,Aadc) inmDAneuroblasts and neurons,as well as the expression of Th in mDA neurons (Ferri et al., 2007;Stott et al., 2013). Thus, the two morphogen-controlled genenetworks in the mFP, Wnt1-Lmx1a and Shh-Foxa2, cooperativelyregulate not only mDA specification and neurogenesis, but alsodifferentiation and survival (Fig. 2, beige area).Transcription factors that are expressed in postmitotic mDA

neurons fromE10-10.5 to adult stages and control the acquisition of amature mDA phenotype include Nurr1/Nr4a2 (Zetterström et al.,1996) andPitx3 (Maxwell et al., 2005) (Fig. 3B).Nurr1 is required formDA neuroblast survival and differentiation into TH+ mDA neurons(Zetterström et al., 1997). In Nurr1−/− mice, mDA neuroblastsbecome PITX3+ but fail to survive and are gradually lost (Le et al.,1999; Saucedo-Cardenas et al., 1998). In agreement with this,NURR1 regulates the expression of several genes that define amaturemDA neuron, including Th, Slc18a2/Vmat2 (solute carrier family-18member-2/vesicular monoamine transporter-2), Slc6a3/Dat (solutecarrier family-6 member-3/dopamine transporter), Ddc/Aadc, Ret(c-ret proto-oncogene), Bdnf (brain-derived neurotrophic factor) andCdkn1c (cyclin-dependent kinase inhibitor 1C) (Gil et al., 2007;Jankovic et al., 2005; Joseph et al., 2003; Saucedo-Cardenas et al.,1998; Smits et al., 2003; Wallen et al., 2001; Volpicelli et al., 2007;Zetterström et al., 1997) (Fig. 3B; Table 1).Pitx3 is one of the earliestmarkers of mDA neurons (Chung et al., 2005b; Jacobs et al., 2007;Nunes et al., 2003; Semina et al., 1997; Smidt et al., 1997). UnlikeNurr1, deletion of Pitx3 does not change the number of VTA/A10TH+ mDA neurons. However the number of SNc/A9 neuronsprogressively decreases in Pitx3−/−mice and is dramatically reducedby birth, indicating that Pitx3 is required for the survival of SNc/A9mDAneurons (Hwang et al., 2003;Maxwell et al., 2005; Smidt et al.,2004; Smidt and Burbach, 2007; van den Munckhof et al., 2003;Yang et al., 2013b). One direct target of PITX3 is the Aldh1a1 gene(aldehyde dehydrogenase-1a1, or Ahd2), which increases retinoicacid and upregulates Th and Drd2 (dopamine receptor 2) butdecreasesDlk1 (delta-like 1 homolog) in anterior mDA cells (Jacobset al., 2011). PITX3 also works independently of retinoic acid toupregulate Vmat and DAT and downregulate Cck (cholecystokinin)and En1/2 (Jacobs et al., 2011; Veenvliet et al., 2013) (Table 1).Moreover, Nurr1 and Pitx3 regulate each other (Jacobs et al., 2009;Volpicelli et al., 2012) and are required for the maintenance ofadult mDA neurons (Kadkhodaei et al., 2009; van den Munckhofet al., 2003). Interestingly, Nurr1 also regulates En1 (Sousa et al.,2007), which in turn regulates Pitx3, Aldh1a1, Th, Slc18a2/Vmat2,Slc6a3/Dat, Cck and Nts contributing to the proper induction ofdistinct mDA subsets (Veenvliet et al., 2013) (Table 1). Moreover,EN1/2 enhances the translation of mitochondrial complex I subunitsand promotes the survival of adult mDA neurons in models of PD

in vivo (Alvarez-Fischer et al., 2011). In sum, NURR1, PITX3 andEN1/2 are crucial regulators of terminal differentiation, as well assurvival and maintenance of mDA neurons (Fig. 2, beige area).

The survival of mDA neurons in vitro and in animal models ofPD is regulated by several families of neurotrophic factors,including the transforming growth factor family (TGFβ2/3)(Poulsen et al., 1994; Roussa et al., 2009); members of theneurotrophin family, such as brain-derived neurotrophic factor(BDNF) (Frim et al., 1994; Hyman et al., 1991); glial cell-line-derived neurotrophic factor (GDNF) (Akerud et al., 2001; Arenaset al., 1995; Beck et al., 1995; Choi-Lundberg et al., 1997; Gashet al., 1996; Kordower et al., 2000; Lin et al., 1993; Rosenblad et al.,1998; Tomac et al., 1995); other members of the GDNF family,such as neurturin or persephin (Akerud et al., 1999, 2002; Horgeret al., 1998; Rosenblad et al., 1999, 2000); and a novel familyformed by mesencephalic astrocyte-derived neurotrophic factor(MANF) and conserved dopamine neurotrophic factor (CDNF orArmetl1) (Lindholm et al., 2007; Petrova et al., 2003; Voutilainenet al., 2009).

Embryonic deletion of Tgfβ3 (Zhang et al., 2007), but not Bdnf orGdnf (Baquet et al., 2005; Moore et al., 1996; Pichel et al., 1996;Sanchez et al., 1996), reduced the numbers of mDA neurons.However, conditional deletion of Gdnf in adult mice (Gdnff/f;Cre-ERTM) causes mDA neuron loss (Pascual et al., 2008), indicatingthat GDNF is subsequently required to maintain the survival ofmDA neurons. Interestingly, members of the TGFβ family arerequired for GDNF to exert its effects (Krieglstein et al., 1998), andthe expression of the GDNF receptor c-ret (Trupp et al., 1996) ismaintained byNurr1 (Decressac et al., 2012). Moreover, GDNF hasbeen suggested to regulate Bdnf expression via a Gdnf-Pitx3-Bdnftrophic loop (Peng et al., 2011). Thus, multiple lines of evidenceindicate that networks formed by morphogens, transcription factorsand neurotrophic factors promote the differentiation and survival ofmDA neurons.

Generating human mDA neurons in vitroOur ability to recreate human mDA neurons in vitro has built in largepart on the growing knowledge of how these cells develop in vivo andhas opened up unprecedented opportunities in disease modeling,drug discovery and cell replacement therapies for PD. Multiplecandidate cell sources are now available to generate DA neurons thatare potentially suitable for therapeutic applications (Fig. 4). Theseinclude endogenous fetal NS/PCs, PSCs and adult somatic cells,which can be used for reprogramming (Fig. 4A-D). However, not allprotocols are equally efficient. Notably, NS/PCs expanded withmitogens as monolayers (Sanchez-Pernaute et al., 2001) orneurospheres (Maciaczyk et al., 2008; Riaz et al., 2002; Ribeiroet al., 2012), or when immortalized (Castelo-Branco et al., 2006;Courtois et al., 2010; Ramos-Moreno et al., 2012; Wagner et al.,1999), have shown variable numbers and quality of DA neurons(Fig. 5A-E), probably due to heterogeneity and/or late developmentalstage of the cells and the protocols used. More recently, long-term-neuroepithelial stem (lt-NES) cells have emerged as a morehomogeneous early stage population that can be derived fromeither human fetal hindbrain tissue (Tailor et al., 2013) or PSCs,which can be differentiated into TH+ neurons (Falk et al., 2012; Kochet al., 2009) (Fig. 5F). However, it remains to be determined whetherthese cells could generate correctly specified mDA neurons withmore advanced differentiation protocols. In this section, we will firstfocus on human PSCs, which can be correctly specified into mDAneurons that are functional in vivo, and are thus the closest to apotential clinical application; and second, on somatic cell

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reprogramming, a novel and promising strategy to generate DAneurons.

Directed differentiation of PSCs into mDA neuronsHuman PSCs (hPSCs), whether derived from the inner cell mass ofan embryo at the blastocyst stage (embryonic stem cells, ESCs)(Thomson et al., 1998), or generated via reprogramming ofdifferentiated somatic cells (induced pluripotent stem cells,iPSCs) (Takahashi et al., 2007), are an attractive cell source forregenerative medicine, given their capacity to self-renew and togenerate all the cell types in an organism. Numerous protocolsdescribing the differentiation of mDA neurons from PSCs have beendeveloped during the last decade. Initial approaches to generatehuman DA neurons were based on adaptations of mouse NS/PC andESC protocols, which relied on the generation of embryoid bodies,the use of stromal cells or astrocytes as feeders, and the activation ofa few key signaling pathways (SHH, FGF8, NURR1) to recapitulatesome aspects of mouse embryonic development (Kawasaki et al.,2000; Kim et al., 2002; Lee et al., 2000; Wagner et al., 1999). Earlyhuman ESC differentiation protocols produced high numbers ofTH+ neurons (Park et al., 2005; Perrier et al., 2004; Roy et al., 2006;Zeng et al., 2004), but none of them generated cells co-expressingtwo transcription factors required for proper mDA neuronspecification, FOXA2 and LMX1A. Early protocols resulted inincorrectly specified TH+ cells (Perrier et al., 2004) that, although

capable of releasing dopamine, survived poorly after transplantation(Park et al., 2005), and could overgrow and generate undesirableprogeny (Roy et al., 2006; Zeng et al., 2004).

Oneof the first protocols to induce correctly specifiedmDAneuronsfrom human ESCs (hESCs) was based on the forced expression ofLMX1A in hESCs treated with SHH and FGF8 (Friling et al., 2009).The resulting cultures contained 50%TH+ neurons,most ofwhich co-expressedmDAmarkers, such as LMX1B,NURR1, PITX3 andDAT(Fig. 5G). Moreover, very few serotonin or motor neurons weredetected in the cultures, suggesting selective mDA specification. Asimilar protocol produced human mDA neurons that releaseddopamine, exhibited appropriate electrophysiological properties andsurvived intracerebral transplantation for up to 5 months (Sanchez-Danes et al., 2012a). However, the in vivo functionality of humanLMX1A-overexpressing cells in animal models of PD was notexamined.

A more rigorous temporal implementation and recapitulation ofmorphogenic signals important for mDA neuron developmentimproved the differentiation protocols. The use of dual SMADinhibitors at the beginning of the differentiation protocol, to inhibitBMP, nodal, activin and TGFβ signaling, improved neuralinduction and eliminated the need for feeder layers (Chamberset al., 2009). However, this protocol relied on patterning with SHHand FGF8 and did not result in correctly specified mDA neurons(Fig. 5H). Subsequently, it was found that administration of

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Fig. 4. Regenerative medicine-based strategies for the treatment of Parkinson’s disease. (A) Human fetal ventral midbrain (VM) tissue has been theclassical source of tissue for transplantation and has provided proof of principle for cell replacement therapy in Parkinson’s disease (PD). (B,C) Midbraindopaminergic neurons have also been generated from human PSCs, derived either from blastocysts (ESCs; B) or by reprogramming of fibroblasts (iPSCs; C).Protocols involve following the developmental steps to first generate ectoderm, neural plate (NP) and neuroepithelial cells (NEP), followed by midbrain floor plate(mFP), midbrain dopaminergic (mDA) progenitors (mDP), mDA postmitotic neuroblasts (mDNb) andmDA neurons. (D) An alternativeway to createmDA neuronsis by direct reprogramming of fibroblasts into induced mDA neurons. (E) mDA neurons and iDA cells can be used for transplantation. (F) Direct reprogrammingcould also be performed in vivo, to generate iDA neurons in situ, from host cells in the adult brain, without the need for cell transplantation. (G) mDA neuronsderived from PD patients or with introduced PD mutations can be compared with controls or corrected mutations and used for disease modeling and drugdiscovery.

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Fig. 5. Protocols for the differentiation of human NS/PCs (A-E) and PSCs (F-M) intomDA neurons.Human fetal VM tissue has been used for the expansionand differentiation of mDA progenitors/stem cells into TH+ cells (yellow) or correctly specified mDA neurons (red). These cells have been grown as monolayers(A), neurospheres (B-D) or immortalized cell lines (E). Human PSCs (hPSCs) derived from blastocysts (ESCs) or reprogrammed fibroblasts (iPSCs) have beenused to generate stable long-term neuroepithelial stem (lt-NES) cells (F), embryoid bodies (EB, G), or for direct differentiation into mDA neurons (H-M).References: A, Sanchez-Pernaute et al. (2001); B, Riaz et al. (2002); C, Maciaczyk et al. (2008); D, Ribeiro et al. (2012); E, Courtois et al. (2010); F, Falk et al.(2012); Koch et al. (2009); G, Friling et al. (2009); H, Chambers et al. (2009); I, Kriks et al. (2011); J, Kirkeby et al. (2012); K, Denham et al. (2012); L, Xi et al.(2012); M, Doi et al. (2014). Small molecules, such as GSK3β inhibitors (GSK3i), have allowedmFP induction andmDA specification. SMAD inhibitors (LDN, SB,A83-01) have substituted noggin (NOG). Shh has been substituted by the smoothened agonist (SAG) and/or purmorphamine (Pur). AA, ascorbic acid;DAPT, γ-secretase inhibitor; dbcAMP, dibutyryl cyclic adenosine monophosphate; FK, forskolin. Green lightning symbols in E and G indicate overexpression ofthe indicated factors. * Circled asterisk indicates cells that have shown functionality in animal models of PD.

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high levels of SHH during neural induction was crucial for FPspecification (Fasano et al., 2010). However, correct midbrainspecification was not achieved until WNT/β-catenin signaling, anessential pathway in mDA neuron development (Andersson et al.,2013; Castelo-Branco et al., 2004, 2003; Chung et al., 2009;Joksimovic et al., 2009b; Prakash et al., 2006; Tang et al., 2010;Yang et al., 2013a), was implemented. Indeed, addition of GSK3βinhibitors allowed for the generation of correctly specified hPSC-derived mDA neurons in a reliable and efficient manner. This wasfirst clearly demonstrated by the Studer group (Kriks et al., 2011),who produced cultures with 80% FOXA2+ cells and 75% TH+ cells(Fig. 5I). Further characterization of these cultures revealedabundant expression of genes involved in mDA neurondevelopment and high levels of co-localization of LMX1Aand FOXA2 in DA progenitors and neurons, indicating correctmDA specification. These cells exhibited biochemical andelectrophysiological properties of mature mDA neurons, and,when transplanted in rodent models of PD, they survived, retainedmarker expression, did not form overgrowths and inducedfunctional recovery in a battery of drug-induced and spontaneousmotor-behavioral tests. Subsequently, the Parmar group used asimilar protocol, but involving the formation of embryoid bodies(Kirkeby et al., 2012). This also led to correctly specified andfunctional mDA cells capable of long-term survival withoutovergrowth formation, which innervated target structures andimproved both drug-induced and spontaneous motor behavior in arat unilateral 6-OHDA model of PD, similar to fetal humanmidbrain tissue (Grealish et al., 2014). GSK3β inhibitors have beenalso successfully used in other protocols to produce correctlyspecified mDA neurons (Denham et al., 2012; Xi et al., 2012)(Fig. 5K,L). These protocols varied somewhat in the doses and thestarting day of GSK3β inhibitor treatment, with day 3 mimickingcloser midbrain development. Finally, a more recent study usedlaminin 511-E8 to promote adhesion of hPS-derived cells duringmFP induction, followed by differentiation as floating spheres(Fig. 5M) (Doi et al., 2014). These protocols lead to correctlyspecified mDA neurons that survived transplantation and improveddrug-induced circling behavior.These recent reports emphasize the importance of implementing

development-based protocols to recapitulate the in vivo exposure tomorphogens such as WNT1 and SHH. In this context, it should benoted that GSK3β inhibitors have off-target effects (Bain et al.,2007) and that GSK3β regulates pathways other than WNT/β-catenin (Hur and Zhou, 2010). Moreover, activating WNT/β-catenin signaling in dopaminergic cells does not always lead tomDA differentiation, as found in the case of WNT3A (Anderssonet al., 2013; Castelo-Branco et al., 2003). Future experiments shouldthus focus on examining whether other pathways are activated andthe extent to which these are required for the observed effects.Furthermore, the need for other factors in WNT/β-catenin-activatedcultures should be reassessed. For instance, in the absence ofGSK3β inhibitors, a period of FGF/ERK signaling blockadefollowed by FGF8 treatment upregulated Wnt1 and promotedmDA differentiation of ESCs (Jaeger et al., 2011). However, inthe presence of GSK3β inhibitors, FGF8 was not required formDA differentiation of ESCs (Kirkeby et al., 2012). In sum,understanding the precise effect of and requirement for differentsignaling molecules is essential in order to further refine currentprotocols, to develop cell replacement therapies and to selectivelygenerate mDA neurons of the SNc/A9 subtype (Box 2), the cells thatimprove functional outcomes in animal models of PD (Grealishet al., 2010).

Direct reprogramming of somatic cells into mDA neuronsThe discovery that mature somatic cells such as fibroblasts can bereprogrammed into iPSCs (Takahashi and Yamanaka, 2006) orother somatic cell types (Davis et al., 1987) by forced expression oftranscription factors has revolutionized regenerative medicine, as itindicates that we might be able to generate any cell type from amature somatic cell. Direct reprogramming relies on our capacity todirectly (by forced expression) or indirectly (i.e. via microRNAs andsmall molecules) regulate the activity of lineage-specifictranscription factors that reassign cell fate (Ladewig et al., 2013).To date, numerous somatic cell types have been generated via directreprogramming, including induced DA (iDA) neurons that havebeen used for transplantation (Caiazzo et al., 2011; Dell’Anno et al.,2014; Kim et al., 2011; Liu et al., 2012b; Pfisterer et al., 2011;Torper et al., 2013) (Fig. 4D,E).

In vitro, induced neurons (iNs) have been obtained by forcedexpression of pan-neuronal factors in cells derived from the threegerminal layers of the embryo (Fig. 6A). The most common factorsused to reprogram fibroblasts (Vierbuchen et al., 2010), hepatocytes(Marro et al., 2011) orastrocytes (Torperet al., 2013) into iNsareBrn1,Ascl1 andMyt1l (BAM). By contrast, the reprogramming of iDA cells(Fig. 6B) was achieved using midbrain-specific factors, such asLmx1a,Ascl1 andNurr1 (LAN) alone (Caiazzo et al., 2011) or togetherwith additional factors, such as FoxA2, En1 and Pitx3 (Kim et al.,2011). In both cases, the resulting iDA cells showed expressionprofiles resembling more closely those of mDA neurons than offibroblasts. iDA cells expressed endogenous Lmx1a (Caiazzo et al.,2011) or endogenous Nurr1 and Pitx3 as well as eGFP from theendogenous Pitx3 locus (Kim et al., 2011). Furthermore, the rodent-

Box 2. DA neuron subpopulationsmDA neurons are traditionally classified by their anatomical position,projection field, function and marker expression. SNc/A9 DA neuronsprimarily express Aldh1a1 andGirk2/KCNJ6 (G protein-regulated inwardrectifier K+ channel subfamily-J member-6), whereas VTA/A10 neuronspredominantly express the calcium-binding proteins Calb1, Calb2(calbindin 1 and 2) and Cck (cholecystokinin) (Veenvliet et al., 2013).Recent studies have shown that Otx2 is predominantly expressed in

VTA/A10 neurons in the developing and adult brain (Chung et al., 2005a;Di Salvio et al., 2010a,b) and is selectively required for the specification(Di Salvio et al., 2010a) and neurogenesis (Di Giovannantonio et al.,2013) of VTA/A10 neurons. Similarly,Wnt1 hypomorphic mice (swaying,Wnt1SW/SW) exhibit a complete loss of VTA, but not SNcmDA neurons, aphenotype transiently phenocopied by Fgf8 conditional knockout(En1Cre) mice, at E12.5 (Ellisor et al., 2012). On the contrary,conditional deletion of Foxa1 and Foxa2 (DATcre/+;Foxa1/2) showed agreater loss of Girk2 and SNc/A9 than Calb1 and VTA/A10 neurons(Stott et al., 2013). Similarly, deletion of Sox6, a factor predominantlyexpressed in SNc/A9 neurons, reduced the number of TH+/Aldh1a1+

SNc/A9 neurons, while increasing that of TH+/Cb+ VTA/A10 neurons,indicating that Sox6 is required for the specification of A9 neurons(Panman et al., 2014). Thus, whereasOtx2 andWnt1 are predominantlyrequired for the specification of VTA/A10 neurons, Foxa1/2 and Sox6 aremainly necessary for the specification of SNc/A9 neurons. Combined,these results suggest that the balance between the Otx2-Wnt1-Lmx1a/band the Shh-Foxa1/2 pathways (Fig. 2) control mDA neuron subtypespecification.However, our knowledge of the molecular differences between these

cells is still limited, and it is possible that additional genes are ultimatelyresponsible for subtype identity. Interestingly, five molecularly distinctmDA neuron subtypes in mice have been suggested very recently(Poulin et al., 2014). Studies examining unbiased global geneexpression in single human mDA neurons are required to confirm andidentify novel, molecularly defined mDA neuron subtypes.

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Fig. 6. Direct reprogramming of somatic cells into induced neurons and dopamine neurons. Cells from different species (mouse, blue arrows; human, redarrows; both, green arrows) and germ layers (endoderm, blue area; mesoderm, green area; ectoderm, yellow area) have been reprogrammed into neurons.(A) Schematic representation of the protocols used to obtain induced neurons (iNs) in vitro, from hepatocytes, fibroblasts, hematopoietic cells, pericytes andastrocytes. Proteins and small molecules are in parentheses. Factor combinations: BAM (Brn1, Ascl1 andMyt1l); LAN (Lmx1a, Ascl1 andNurr1).References: a,Heins et al. (2002); b, Berninger et al. (2007); Heinrich et al. (2010, 2011); c, Karow et al. (2012); d, Vierbuchen et al. (2010); e, Marro et al. (2011); f, Pfistereret al. (2011); g, Pang et al. (2011); h, Qiang et al. (2011); i, Yoo et al. (2011); j, Ambasudhan et al. (2011); k, Ladewig et al. (2012); l, Xue et al. (2013); m, Torperet al. (2013); n, Guo et al. (2014); o, Castan o et al. (2014). (B) Induced dopamine neurons (iDAs) have been generated in vitro from fibroblasts andastrocytes by different protocols. References: a, Caiazzo et al. (2011); b, Kim et al. (2011); c, Addis et al. (2011); d, Pfisterer et al. (2011); e, Torper et al.(2013); f, Liu et al. (2012b). (C) Induced neurons produced in vivo by reprogramming of endogenous mouse brain cells, such as astrocytes and NG2+ cells in thecerebral cortex (CTX), spinal cord (SC) or striatum (STR) in situ. Whereas cells with glutamatergic (Glu) or GABAergic traits (GABA) have been observed,iDA neurons have not yet been obtained from adult brain cells in vivo. References: a, Grande et al. (2013); b, Torper et al. (2013); c, Niu et al. (2013);d, Su et al. (2014); e, Guo et al. (2014); f, Heinrich et al. (2014). AD, Alzheimer’s disease transgenic model; CHIR, GSK3b inhibitor; NOG, noggin; SB,Smad inhibitor; VPA, valproic acid.

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derived cells exhibited mature DA neuronal properties as definedelectrophysiologically and through dopamine release. Lastly, thesetwo studies showed integration of murine-derived iDA cells into themouse brain. The iDA neurons produced byKim and colleagues wereable to partially alleviate motor deficits in a mouse model of PD, butrequired tenfold more cells than fetal DA tissue grafts, indicating thattheir level of functionality is low (Kim et al., 2011). A slight variationof this protocol, using Lmx1b instead of Lmx1a, also reprogrammedprimary mouse postnatal cortex astrocytes into functional iDA cells,although these have not been tested in vivo (Addis et al., 2011).Notably, the fact that somatic cell types derived from different germlayers, such as fibroblasts and astrocytes, can be reprogrammed intoiDA cells by a similar combination of factors highlights the inherentrobustness of such factors in the reprogramming process.In an alternative strategy, a pan-neuronal identity was first

induced with BAM, before further mDA neuron factors were added,including Lmx1a and Foxa2 alone (Pfisterer et al., 2011) or incombination with Lmx1b andOtx2 (Torper et al., 2013). These cellssuccessfully engrafted in the striatum of a 6-OHDA rat model of PD,but very few cells survived after 6 weeks (Pfisterer et al., 2011),indicating that they do not behave like human fetal or hESC-derivedmDA neurons. In another study, Ascl1, Nurr1, Pitx3 and twoadditional factors, Sox2 and Ngn2, but not Lmx1a, were used (Liuet al., 2012b), although, again, the resulting iDA cells engrafted inthe striatum of 6-OHDA lesioned rats but survived poorly and didnot exhibit a neuronal morphology.More recently, the Broccoli group (Dell’Anno et al., 2014)

demonstrated pan-DA marker expression, functional integrationin vivowithout tumor formation or overgrowths, and partial reductionof motor deficits in an in vivomodel of PD by using mouse iDA cellsreprogrammed with LAN. Expression of DREADDs (designerreceptors exclusively activated by designer drugs) in iDA cells andtreatment with the corresponding ligand further improved firingactivity, dopamine release and behavioral recovery in a model of PD.These results represent an important milestone in the field as theefficiency of iDA cells was comparable to that of embryonic mDAneuron grafts. In the future, it will be important to perform similarstudies with human iDA cells and examine their in vivo long-termexpression of midbrain-specific markers.Direct in vivo reprogramming of in situ adult mouse brain cells into

neurons has become a reality in recent years (Fig. 4F), although iDAcells have not yet been obtained in vivo. Neurons have beenreprogrammed from other types of neurons (De la Rossa et al.,2013; Rouaux and Arlotta, 2013), unidentified non-neuronal cells(Grande et al., 2013) (Fig. 6Ca), astrocytes (Guo et al., 2014;Niu et al.,2013; Su et al., 2014; Torper et al., 2013) (Fig. 6Cb-e), andNG2+ cells(Guo et al., 2014; Heinrich et al., 2014) (Fig. 6Ce,f) have beenreprogrammed into neurons in vivo. These neurons have shownmatureelectrophysiological properties, but their neuronal phenotypes areoften incomplete and their numbers are low. Further developments arethus necessary to develop this method for cell replacement therapy forPD.Notably, injury promoted (Grande et al., 2013;Guoet al., 2014) orwas required (Heinrich et al., 2014) for neuronal reprogramming invivo, indicating that this strategy is particularly well suited for neuralrepair. Indeed, the reprogramming of glial cells into neurons wouldallow neurons lost by disease to be replaced,while reducing the excessof reactive glia, and regaining balance between glia and neurons inneurodegenerative diseases.

The induction of iDA cells: a regulatory logic?The various combinations of reprogramming factors used togenerate iDA neurons in vitro (Fig. 6B) all combine Ascl1 with

additional midbrain- and DA neuron-specific transcription factorgenes. As Ascl1 alone is sufficient to convert mouse fibroblasts intoiN cells (Chanda et al., 2014; Vierbuchen et al., 2010), but is notrequired for mDA neuron development (Kele et al., 2006), Ascl1 islikely to play a generic neurogenic or neuronal reprogramming roleand has been proposed to act as a pioneer factor (Wapinski et al.,2013). Another gene widely used for the induction of mDA fate isLmx1a, a known fate determinant required for mDA neurondevelopment (Andersson et al., 2006b; Deng et al., 2011), which isessential for mDA neuron programming and a core component indirect iDA reprogramming. Other midbrain transcription factorgenes, such asNurr1, Foxa2 and Pitx3, appear dispensable as singlegenes in some of the protocols, but in combination with Ascl1, andat least one other mDA transcription factor, contribute to improveiDA reprogramming. Thus, currently available data suggest a modelin which the acquisition of an iDA phenotype requires thecombination of the pioneer factor Ascl1, Lmx1a as a core iDAreprogramming factor, and at least one or more mDA neurontranscription factor, as helpers in iDA reprogramming. Combined,these results indicate that iDA reprogramming follows basicdevelopmental principles and emphasizes the importance ofunderstanding the mechanism of action of developmental factorsin the context of reprogramming. In the future, it will be important tocharacterize the transcriptional networks and phenotype of iDAcells in order to determine whether a mature mDA-like iDA cell canbe generated, whether different protocols lead to similar or distinctiDA cells and whether different cells correspond to separate routesand/or stages of reprogramming.

Conclusions, perspectives and future directionsOur knowledge of the developmental mechanisms that regulatemDAneuron development, aswell as the tools currently available forgenerating mDA neurons (cells, factors and protocols), have grownexponentially in the last few years. These developments havegenerated optimism in the regenerative medicine field with regard tothe future development of cell replacement therapy and drugdiscovery for PD. However, issues important for mDA neurondevelopment and phenotype stability, such as genetic and epigeneticregulatory mechanisms or the strength of signaling, are still poorlyunderstood.

Reprogrammed iDA cells for cell transplantationAs discussed above, direct reprogramming is currently emerging as apossible alternative to stem cell-derived mDA neurons for cellreplacement therapy. The advantages of such amethodwould be that itcould avoid the generation of proliferative immature cells, reducing therisk of tumor/outgrowth formation, and autologous cells would notrequire immunosuppression. However, it remains to be determinedwhether the phenotype and functionality of directly reprogrammedhuman iDA cells is the same as human mDA neurons derived fromother sources, and whether transplanted human iDA cells aresufficiently stable and safe to be used for long-term correctionof functional and behavioral deficits in models of PD. Progress in thegeneration of iDA cells will thus require: (1) A better understandingand ability to optimize the iDA reprogramming mechanisms,potentially by exploiting novel microRNAs or small molecules;(2) a detailed characterization of the iDA phenotypes generatedby distinct reprogramming factor combinations, and of phenotypestability compared with endogenous DA cells; (3) determining thesafety of the method with regard to insertional mutagenesis, partialreprogramming, proliferation and host inflammatory/immuneresponse; (4) for in vivo iDA reprogramming, the development of

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cell type-specific efficient reprogramming methods to selectivelytarget the desired cell type whilst avoiding possible damage to othercells. So far, no iDA cells have been produced from endogenous adultbrain cells in vivo, and selective targeting has been achieved by usingCre-recombinase or tetracycline transactivator (tTA) driver mouselines. Viruses or carriers capable of selectively, efficiently and safelytargeting specific cell types will be necessary in the future.

Towards cell replacement therapies for PD with PSCsRecent protocols for generating mDA neurons from hPSCs offer thepossibility of generating an unlimited number of safe and functionalcells in vivo (Kriks et al., 2011) that survive for up to 6 months. Inaddition, they are capable of innervating distant target structures(Grealish et al., 2014) and modulating the glutamatergic input tostriatal medium spiny neurons (Steinbeck et al., 2015). This has beenmade possible because of an improved understanding ofmDA neurondevelopment anda focuson studyinghuman cells, overcoming speciesdifferences and allowing the preclinical development of stem cell-based cell replacement therapy for PD. Future challenges includeimproving our understanding of mDA neuron subtype specificationand developing more effective protocols to generate human SNc/A9mDA neurons. Strategies to improve protocols might involve cellsorting (Box 3), the incorporation of novel developmental factors tocurrent differentiation protocols, as well as achieving the rightsignaling level or balance between different factors and pathways(Box 2; Fig. 2). Shorter protocols, for example starting from hPSC-derived mDA progenitors, would also facilitate the development ofclinical applications by saving time and costs. All these protocols willneed to be upscalable, use defined human components and bethoroughly tested in order to be good manufacturing practice (GMP)compliant and thus useful for cell replacement therapy in the clinic.Moreover, the functionality of GMP-produced hPSC-derived mDAneurons should also be examined in animal models of PD. For use inreplacement therapy, cells must meet a number of criteria, includingthe ability to (1) not only survive transplantation, but integratewithoutexcessive growth; (2) acquire a stable mature phenotype in vivo withthe loss of immature markers in long-term surviving grafts; (3)

re-innervate the host striatum, but no other targets; (4) releasedopamine and exhibit appropriate electrophysiological properties; (5)induce functional recovery in sensorimotor behavioral tests relevant toPD; and (6) attain anhomogeneous populationof highly efficient SNc/A9 cells in order to reduce the number of cells required fortransplantation. Finally, transplantation in primates might be usefulin order to prepare clinical trials and ascertain the degree of fiberoutgrowth and functional capacity of the cells, as well as issues ofimmunogenicity in the caseof allogeneic orautologous iPSC/iNwork.

The challenges facing cell replacement therapy in PD are greatbecause new therapies need to be competitive against existingtherapies, such as L-DOPA or DBS. The experience from successfulopen label trials using fetal hVM tissue suggests that cell replacementcan be a competitive therapy. However, in order to further develop thisstrategy, new double-blind fetal tissue clinical trials with improvedstructure are needed (Evans et al., 2012). Future trials will alsoprobably involve the use of hPSCs, as they are more amenable andupscalable sources of mDA neurons. hESCs are currently consideredsafer than iPSCs, as they do not require genetic modification.However, iPSCs can be generatedwith safer non-integrative methods,such as RNAor protein transduction or smallmolecules (Hargus et al.,2010; Kiskinis and Eggan, 2010; Sundberg et al., 2013). Theadvantage of iPSCs is that they can be derived from readily accessibleautologous somatic cells, which is ethically less controversial.Moreover, PD-iPSCs would not require immunosuppression, buttheymight carry genetic or environmental damage, predisposing themto the development of PD. Avoiding this problem would involvecorrection of the cells or the use of immunosuppressionwith hESCs orhiPSCs derived from healthy individuals. Either way, the creation ofhPSC banks covering the main human leukocyte antigens (HLAs)might allow treatment of multiple patients.

Reprogrammed cells as tools for disease modeling and drugdiscoveryPD iPSC-derived mDA, but not PD iDA cells, have been so far usedfor PD modeling. The main advantage of human PD iPSC-derivedmDA cells compared with non-reprogramming strategies is that

Box 3. Selection of mDA cells for transplantationIn many cases, differentiation and reprogramming protocols generateheterogeneous populations of cells. Fluorescence-activated cell sorting(FACS) can be used both to remove undesired cell types and to enrich formDA neurons or subtypes thereof. This approach has been used toselect for neural fate, by using markers such as the glycoprotein CD133(prominin 1, PROM1) and NCAM1 (neural cell adhesion molecule 1, orCD56) (Pruszak et al., 2007). For transplantation studies (Fig. 4E),committed, but not fully differentiated mDA rodent cells (Jönsson et al.,2009; Villaescusa and Arenas, 2010) at a NURR1+ neuroblast stage(Ganat et al., 2012), have been found to perform best.

More recently, NCAM+CD29low (CD29 for integrin beta 1 or ITGB1)cells were sorted from a population of human PSCs differentiated withthe midbrain floor plate protocol (Sundberg et al., 2013) to remove non-neural tumorigenic cells and enrich for FOXA2, LMX1A and TH+

neurons. The transplantation of these cells led to improved behavioralrecovery in an animal model of PD (Sundberg et al., 2013). Similarly,human iPSCs sorted with CORIN and expressing the mDA markersFOXA2 and LMX1A survived grafting in 6-OHDA lesioned rats andinduced behavioral recovery without tumor formation (Doi et al., 2014).However, sorting protocols that enrich for SNc/A9 cells have yet to bedeveloped, and it is also important to eliminate cell types such asserotonergic cells that might cause side graft-induced dyskinesias(Carlsson et al., 2009, 2007; Politis et al., 2010).

Box 4. Generation of mDA cells for in vitro PDmodelingWith the advent of reprogramming technologies, PD-specific iDA cells andiPSC-derived DA neurons have become very attractive options for PDmodeling (Fig. 4G). Thus far, studies have focused on PD-iPSCs carryingLRRK2mutations (Cooperet al., 2012; Liu et al., 2012a;Nguyenet al., 2011;Reinhardt et al., 2013; Sanchez-Danes et al., 2012b; Sanders et al., 2014),SNCA triplication(Byersetal.,2011;Devineetal., 2011),SNCAA53Tmutation(Ryanet al., 2013;Soldneret al., 2011),PINKmutations (Cooperet al., 2012;Seibleret al., 2011) orGBA1heterozygosity (Mazzulli et al., 2011; Schöndorfet al., 2014). Most of these studies relied on protocols with suboptimalWNT/β-catenin activation, which do not give rise to correctly specified mDAneurons. However, those studies using sufficient WNT/β-catenin activationled to cell preparations with variable percentages of mDA neurons, rangingfrom 20% (Schöndorf et al., 2014) to 70% (Ryan et al., 2013).As alterations in PD are cell-type specific and context dependent, it is

necessary to further improve current mDA differentiation protocols for PD-iPSCs. Key considerations include the generation of control and PD-iPScellswith identical geneticbackgrounds (Hockemeyeretal., 2009;Maetzelet al., 2014;Wanget al., 2013; Zouet al., 2009), improvingyieldof correctlyspecified and functional SNc/A9 neurons, reducing the culture time bystarting from stable intermediates (e.g. mDA progenitors) and developinghigh content assays, in which the cells of interest and their phenotypes areexamined at a single-cell level at an appropriate spatial and temporalresolution (Jain and Heutink, 2010; Xia and Wong, 2012).

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modeling is performed in human cells directly derived from PDpatients (Box 4). This allows the study of features related to thegenetic background of the patients, such as mutations, haplotypes orpolymorphisms associated with the development of certain typesand features of PD. Another advantage of reprogrammed cells isthat, compared with postmortem samples, they can capture earlystages of the disease, which might be crucial for the development oftherapies aimed at preventing neurodegeneration. Reprogrammedcells can be used to generate other cell types affected in PD or evencreate neural organoids (Kadoshima et al., 2013; Lancaster et al.,2013), which have opened up the possibility of modeling featuresrelated to the anatomical or functional organization of cells in atissue. So far, correctly specified PD-iPS-derived mDA neuronshave been successfully used to model oxidative and nitrosativestress (Ryan et al., 2013), as well as autophagic defects in PD(Schöndorf et al., 2014). PD-iPSCs are thus considered the cell typeof choice for high-throughput screening (HTPS) and drugdevelopment.Gene editing technologies, such as zinc-finger nucleases,

TALENS or the CRISPR/Cas9 system, are currently being used tocorrect or introduce PD mutations and control for the geneticbackground of PD-iPSCs (Chung et al., 2013; Liu et al., 2012a;Reinhardt et al., 2013; Ryan et al., 2013; Sanders et al., 2014;Schöndorf et al., 2014; Soldner et al., 2011). These methods can beused to introduce/correct multiple mutations and examine epistaticinteractions between PD genes, allowing more complete iPSCmodels in which several features of PD are simultaneouslyexamined. In sum, multiple developments in this fascinating areaof research are likely to improve in vitro modeling and drugdiscovery for PD, which, in turn, should contribute to noveltherapeutic interventions.

AcknowledgementsWe would like to thank Roger Barker and members of the Arenas lab for criticalreading of the manuscript.

Competing interestsThe authors declare no competing or financial interests.

FundingThis work was supported by grants from the Swedish Foundation for StrategicResearch (CEDB and SRL Program), the Swedish Research Council [VR2008:2811and 3287, VR2011:3116 and DBRM], Karolinska Institutet (SFO, Thematic Centerin Stem Cells and Regenerative Medicine), the European Commission[NeuroStemCellRepair: HEALTH-F4-2013-602278 and DDPDGENES: HEALTH-2011-278871] and the Czech Ministry of Education and European RegionalDevelopment Fund [KI-MU; CZ.1.07/2.3.00/20.0180].

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